All mammals possess a circadian clock that governs daily oscillations with neural, hormonal, and metabolic functions for the organism with a near 24-h period. The suprachiasmatic nucleus (SCN) within the brain's hypothalamus houses this clock. The SCN has neural connections with various regions of the brain. Prominent among these connections are cholinergic projections from regions of the brain responsible for asleep and alerting, namely the basal forebrain (BF) and brainstem. A considerable body of work is available regarding intracellular signal transduction pathways within SCN. Pharmacological studies in vitro have implicated the M1-subtype of the muscarinic acetylcholine receptor (M1AChR) as the receptor mediating the effects of cholinergics on the circadian system. Recently, the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification has questioned the specificity of the drugs used to implicate the M1AChR in the cholinergic signaling cascade. In lieu of specific pharmacology, other methods must be employed to confidently identify the receptor(s) mediating cholinergic circadian signaling. In this proposal, electrophysiological, behavioral and anatomical studies will be carried out in mice devoid of the M1AChR (M1KO). First, the B6129PF1/J mouse, a mouse of similar genetic background to the M1K0, will be tested for sensitivity to the cholinergic agonist, carbachol, both in vitro and in vivo. Second, the M1KO will be tested for decreased sensitivity to carbachol. Finally, double-label fluorescence confocal microscopy will be used to evaluate whether the cholinergic terminals within the SCN are juxtaposed to M1AChR-containing cells and to map their distribution.